Cell Cycle
Living organisms are composed of cells, whose growth and division require a regular sequence of events and processes that make up a cell cycle. A cell cycle comprises two periods: 1) interphase, the period of cell growth, and 2) mitosis, cell division and the separation of daughter of cells. Some cell cycle events are continuous (e.g., synthesis of RNA, proteins, and lipids), whereas others are discontinuous (e.g., DNA synthesis). Two discontinuous processes for cell survival are the replication of DNA and the segregation of chromosomes to the daughters of cell division during mitosis. If either of these steps are performed inaccurately, the daughter cells will be different from each other and will almost certainly be flawed. Chromosome replication occurs in eukaryotic cells only during interphase; and DNA replication and DNA segregation are mutually exclusive processes.
Interphase is subdivided into the S phase (synthetic phase) when DNA replication occurs, and the gaps, G1 and G2, separating the S phase from mitosis. G1 is the gap after mitosis, before DNA synthesis starts; G2 is the gap after DNA synthesis is complete, before mitosis and cell division. During G1 and G2, no net synthesis of DNA occurs. As mentioned above, during interphase, there is continued cellular growth and continued synthesis of other cellular macromolecules such as RNA, proteins, and membranes. Additionally, centrioles which are composed of microtubules duplicate during interphase.
Segregation of chromosomes as well as mitotic spindles, occurs during mitotic (M) period, normally a relatively brief period in the cell cycle, which culminates in the highly visible act of cell division (e.g., cytokinesis). The appearance of chromosomes as thin threads inside the nucleus indicates the beginning of mitosis. The mitotic period is divided into four substages which are prophase, metaphase, anaphase, and telophase.
During prophase, each chromosome is composed of two chromatids held together by their centromeres. Each of the chromatids contains one of the two daughter DNA molecules replicated during the S phase. The centrioles generate microtubules and move to opposite poles. Those microtubules that associate with fibers and proteins form spindle fibers. At the end of prophase, the centrioles are at opposite poles. Some spindle fibers extend from the centrioles at the poles to the equator of the cell, while others extend from the poles to the chromatids attached to the kinetochores near the centromeres of the chromatids.
In metaphase, the chromosomes move to the equator of the cell and align in the equatorial plane.
During anaphase, the daughter chromatids separate and move toward the pole to which it is linked by a spindle fiber. At the same time, both the cell and the spindle fibers elongate. At late anaphase, a cleavage furrow starts to form which begins the process of cytokinesis.
During telophase, new membranes form around the daughter nuclei. At the end of telophase, cytokinesis is nearly complete; the spindle fibers disappear and the microtubules and other fibers depolymerizes. At the end of the mitotic phase, identical copies of the cellular DNA are distributed to each of the daughter cells.
The Mitotic Spindle and Tubulin
Microtubule formation is important for cell mitosis, cell locomotion, and the movement of highly specialized cell structures such as cilia and flagella. The mitotic spindle is a self-organizing structure that is constructed primarily from microtubules. Among the most important spindle microtubules are those that bind to kinetochores and form the fibers along which chromosomes move. These microtubules are comprised of α-β tubulin dimers. γ-tubulin is a phylogenetically conserved component of microtubule-organizing centers that is essential for viability and microtubule function (T. Horio et al. (1994) J. Cell Biol. 126(6): 1465-73). It is exclusively localized at the spindle poles (also known as spindle pole bodies, SPB) in mitotic animal cells, where it is required for microtubule nucleation (M. A. Martin et al. (1997) J. Cell Sci. 110(5): 623-33; I. Lajoie-Mazenc et al. (1994) J. Cell Sci. 107(10): 2825-37). γ-tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB (R. Ding et al. (1997) Mol. Biol. Cell 8(8): 1461-79).
One protein linked with the mitotic spindle is p53, which is a critical participant in a signal transduction pathway that mediates either a G1 arrest or apoptosis in response to DNA damage (S. E. Morgan et al. (1997) Adv. Cancer Res. 71: 1-25). Loss of p53, in addition to suppression of apoptosis by bcl-2-related genes, may act cooperatively to contribute to genetic instability (A. J. Minn et al. (1996) Genes Dev. 10(2): 2621-31). The oncoprotein, Bcl-2, also has been demonstrated to be cell cycle specific, appearing in early prophase or late G2 and persisting throughout mitosis. The pattern of bcl-2 protein localization shows a diffuse nuclear distribution before chromosome condensation, followed by a specific concentration of bcl-2 at the margins of condensed chromosomes in prophase, metaphase and anaphase (M. C. Willingham et al. (1994) J. Histochem. Cytochem. 42(4): 441-50).
As microtubules and microtubule-related structures are intimately involved in the mitotic process, they have provided a convenient target for putative anti-mitotic compounds. Indeed, microtubules have proven to be extremely labile structures that are sensitive to a variety of chemically unrelated anti-mitotic drugs. For example, colchicine and nocadazole are anti-mitotic drugs that bind tubulin and inhibit tubulin polymerization (Stryer (1988) Biochemistry). When used alone or in combination with other therapeutic drugs, colchicine has been used to treat cancer (WO9303729; J03240726-A), alter neuromuscular function, change blood pressure, increase sensitivity to compounds affecting sympathetic neuron function, depress respiration, and relieve gout (Physician's Desk Reference, (1993) 47: 1487).
Taxol and the vinca alkaloids are chemotherapeutics that bind microtubles. They perturb kinetochore-microtubule attachment and disrupt chromosome segregation. This activates a check point pathway that delays cell cycle progression and induces programmed cell death (P. K. Sorger et al. (1997) Curr. Opin. Cell. Biol. 9(6): 807-14; C. M. Ireland et al. (1995) Biochem. Pharmacol. 49(10): 1491-99). Taxol has been demonstrated to induce tubulin polymerization and mitotic arrest which is followed by apoptosis. Overexpression of Bcl-x(L) in taxol induced cells has been demonstrated to interfere with the activation of a key protease involved in apoptosis (A. M. Ibrado et al. (1996) Cell Growth Differ. 7(8): 1087-94).
Cyclin, Cyclin-Dependent Kinases, and their Inhibitors
Regulation of the cell cycle by cellular constituents ensures the controlled generation of cells with specialized functions. Cyclin and cyclin-dependent kinases (CDKs) are molecules that play a key role in regulating the eukaryotic cell cycle. Cyclin/CDK complexes are formed via the association of a regulatory cyclin subunit (such as cyclin A) and a catalytic kinase subunit (such as cdc2 or CDK1). Sequential formation, activation, and subsequent inactivation of a series of cyclin/cyclin dependent kinase complexes controls the progression of eukaryotic cells through the three phases of the growth cycle (G1, S, and G2) leading to division in the mitotic phase (M). Each step in the cell cycle is regulated by a distinct and specific cyclin-dependent kinase. For example, complexes of Cdk4 and D-type cyclins govern the early G1 phase of the cell cycle, while the activity of the CDK2/cyclin E complex is rate limiting for the G1 to S-phase transition. The CDK2/cyclin A kinase is required for the progression through S-phase and the cdc2/cyclin B complex controls the entry into M-phase (Sherr (1993) Cell 73: 1059-1065).
Cdc2, the first identified CDK, was discovered as a gene essential for both G1/S and G2/M transitions in yeast (Nurse et al., (1981) Nature 292: 558-560). The cloning of the gene encoding the human homolog of Cdc2, CDK1, by complementation led to the identification of cdc2 homologs in all eukaryotes from plants and unicellular organisms to humans and to the realization that cdc2 was only the first member of a family of closely related kinases. CDKs are typical Ser/Thr kinases comprising eleven subdomains shared by all protein kinases. Examples of CDKs include, but are not limited to cdc2, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK7.
Following the discovery of cyclin B in sea urchin eggs, cyclin B homologs were identified in all eukaryotes. Like cdc2, cyclin B is one member of a large family of kinase regulators. Members of the family include but are not limited to cyclin A, cyclin B1-B3, cyclin C, cyclin D1-D3, cyclin E, and cyclin H.
Examples of cyclin/CDK complexes include, but are not limited to cyclin A/cdc2 or cdk2, cyclin B1-B3/cdc2, cyclin C/cdk8, cyclin D1-D3/cdk2, cdk4, cdk5, or ckd6, cyclin E/cdk2, and cyclin H/cdk7.
Cyclin dependent kinase inhibitors (CKIs) are also essential for regulating the cell cycle. CKIs negatively regulate CDK or cyclin/CDK activity by associating with them. By binding specifically to either CDK, or the cyclin/CDK complexes, they inhibit the cyclin/CDK complexes. CKI activity and levels are cell cycle regulated allowing these proteins to function as inhibitors of their cognate cyclin/CDK complexes for very limited periods during the cell cycle. Examples of a few CKIs include purvalanol, olomoucine, roscovitine, flavopiridol, and alsterpaullone.
The discovery that human CDKs, cyclins and CKIs are mutated or abnormally expressed in a number of cancerous cells confirms that these gene products and their functions are essential for mammalian cell cycle regulation (reviewed in Hunter (1993) Cell 75: 839-841; Marx (1993) Science 262: 1644-1645; Marx (1994) Science 263: 319-321; Sherr (1996) Science 274: 1672-1677). Altered expression of cyclins, CDKs, and their modulators in malignant cells, results in deregulated CDK activity and uncontrolled growth of malignant cells.
Apoptosis and Survivin
Apoptosis or programmed cell death is a natural form of death that organisms use to dispose of cells. It occurs in response to different factors such as growth factor addition or withdrawal, antitumoral drugs, viral infections, activation tumor suppressor genes, and cytotoxic agents. These factors are also known to modify cell cycle progression. Evan (1995, Curr. Opin. Cell Biol. 7: 825-834) reports that apoptosis and cell cycle controls are closely linked.
Deregulated expression of inhibitors of apoptosis (programmed cell death) is thought to contribute to cancer by abnormally extending cell viability, favoring the accumulation of mutations, and promoting resistance to therapy (Reed (1999) J. Clin. Oncol. 17: 2941-53). A novel modulator of the cell death/viability balance in cancer was recently identified as survivin (Ambrosini et al. (1997) Nat. Med. 3: 917-21), a member of the Inhibitor of Apoptosis (IAP) gene family (Deveraux et al. (1999) Genes Dev. 13: 239-52).
Survivin is a 16.5 kDa cytoplasmic protein containing a single partially conserved BIR (baculovirus IAP repeats) domain, and a highly charged carboxyl-terminus coiled-coil region instead of a RING finger, which inhibits apoptosis induced by growth factor (IL-3) withdrawal when transferred in B cell precursors (Ambrosini et al. (1997) Nat Med 3: 917-921). Based on overall sequence conservation, the absence of a carboxyl terminus RING finger and the presence of a single, partially conserved, BIR domain, survivin is the most distantly related member of the IAP family, sharing the highest degree of similarity with NAIP (neuronal apoptosis inhibitory protein; Roy et al. (1995) Cell 80: 167-178). Additionally, unlike other IAP proteins, survivin is undetectable in normal adult tissues, but becomes the top fourth transcript expressed in common human cancers (Ambrosini et al. (1997) Nat. Med. 3: 917-21; Velculescu et al. (1999) Nat. Genet. 23: 387-88), such as lung, colon, breast, pancreas, and prostate, and in ˜50% of high-grade non-Hodgkin's lymphomas, in vivo.
Survivin is expressed in the G2/M phase of the cell cycle in a cell cycle-dependent manner, and localized to mitotic spindle microtubules and intercellular actomyosin bridges, i.e. midbodies, during cell division (Li et al. (1998) Nature 396: 580-584). Interference with this topography, or blocking survivin expression, caused increased caspase-3 activity in G2/M (Li et al. (1998) Nature 396: 580-584), and a profound dysregulation of mitotic progression (Li et al. (1999) Nat. Cell Biolog. 1: 461-466), suggesting that survivin may regulate a novel apoptotic checkpoint at cell division. This pathway was dramatically exploited in cancer (Ambrosini et al. (1997) Nat. Med. 3: 917-921), where survivin was identified as one of the top four “transcriptomes” out of 3.5 millions mRNAs, uniformly expressed in cancer, but not in normal tissues (Velculescu et al. (1999) Nat. Genet. 23: 387-388). Additionally, it has been shown that transformed cells are exquisitely sensitive to manipulation at this mitotic checkpoint as interference with survivin expression and function using dominant-negative mutants with point mutations in the conserved baculovirus IAP repeat (BIR) domain or survivin antisense resulted in aberrant mitoses (Li et al. (1999) Nat. Cell Biolog. 1: 461-466) and spontaneous apoptosis (Ambrosini et al. (1998) J Biol Chem. 273: 11177-82; Grossman et al. (1999) Lab Invest 79: 1121; Grossman et al. (1999) J. Invest. Dermatol. 113: 1076-81). This phenotype is unique to survivin and not observed with other apoptosis inhibitors potentially contributing to neoplasia, as antisense inhibition of Bcl-2 increased sensitivity to apoptosis but did not in itself induce cell death (Jansen et al. (1998) Nat. Med. 4: 232).
O'Connor et al. (2000 Proc Natl Acad Sci USA 97: 13103-7) teach that survivin is phosphorylated on Thr34 by the main mitotic kinase kinase complex, cyclin B1/p34cdc2, in vitro and in vivo. Loss of phosphorylation on Thr34 results in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data suggest that survivin is a mitotic substrate of p34cdc2-cyclin B1 and survivin phosphorylation on Thr34 may be required to preserve cell viability at cell division.
Grossman et al. (2001 Proc Natl Acad Sci USA 98: 635-40) report that expression of a phosphorylation-defective survivin mutant (Thr34→Ala) triggered apoptosis in several human melanoma cell lines and enhanced cell death induced by the chemotherapeutic drug cisplatin in vitro. Conditional expression of survivin Thr34→Ala in YUSAC2 melanoma cells prevents tumor formation upon s.c. (subcutaneous) injection into CB.17 severe combined immunodeficient-beige mice. When induced in established melanoma tumors, survivin Thr34→Ala inhibits tumor growth by 60-70% and cause increased apoptosis and reduced proliferation of melanoma cells in vivo.
Anti-cancer treatments exploit activation of cell cycle checkpoints to arrest cell proliferation and induce apoptosis. However, escape mechanisms engendered by tumor cells may preserve cell viability in face of checkpoint activation, favoring aberrant mitotic progression and exacerbating genomic instability. Survivin, a member of the Inhibitor of Apoptosis (IAP) gene family expressed in most human cancers, requires phosphorylation by p34cdc2-cyclin B1 for cytoprotection.
p34cdc2 Survival Checkpoint in Cancer
Checkpoints act as surveillance mechanisms to ensure proper timing of the cell division cycle (C. J. Sherr (1996) Science 274: 1672-77). At mitosis, the assembly of a bipolar spindle is vital to the preservation of genetic fidelity between daughter cells, and is monitored by a checkpoint (A. D. Rudner et al. (1996) Curr. Op. Cell Biol. 8: 773-80) that senses microtubule defects (S. S. L. Andersen (2000) Trends Cell Biol. 10: 261-67), or aberrant kinetochore attachment (R. B. Nicklas (1997) Science 275: 632-637). Activation of the spindle checkpoint by mitotic stresses causes a prolonged arrest of cell division that may eventually lead to apoptosis, or programmed cell death (M. O. Hengartner (2000) Nature 407: 770-76.). This strategy has been exploited for anti-cancer treatments, and agents that perturb microtubule dynamics or interfere with microtubule assembly (P. K. Sorger et al. (1997) Curr. Biol. 9: 807-14) have shown efficacy in the management of common human tumors (Rowinsky and Donehower, 1995).
Among the regulators of apoptosis that may affect the cell death/viability balance of dividing cells, interest has recently focused on the Inhibitor of Apoptosis (IAP) (Q. L. Deveraux, Q. L. et al. (1999) Genes Dev 13: 239-252) protein and mitotic regulator, survivin (D. C. Altieri (2001) Trends Mol. Med. 7: 542-47.). Expressed during cell division in a cell cycle-dependent manner and localized to various components of the mitotic apparatus, survivin has been implicated in both regulation of spindle microtubule function and preservation of cell viability (D. C. Altieri (2001) Trends Mol. Med. 7: 542-47; J. C. Reed, J. C. et al. (2000) Cell 102: 545-48). A critical requisite for survivin function was identified in the phosphorylation on Thr34 (D. S. O'Connor et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13103-107) by the main mitotic kinase, p34cdc2-cyclin B1 (J. Pines (1999) Nat. Cell Biolog. 1: E73-E79). Accordingly, expression of non-phosphorylatable survivin Thr34→Ala prevented phosphorylation of endogenous survivin and triggered apoptosis of various cancer cell types (D. Grossman et al. (2001) Proc. Natl. Acad. Sci. USA 98: 635-640; D. S. O'Connor et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13103-107). When tested in mouse cancer models, expression of survivin Thr34→Ala induced apoptosis in situ, suppressed tumor formation, and inhibited growth of established tumors (D. Grossman et al. (2001) Proc. Natl. Acad. Sci. USA 98: 635-640; M. Mesri et al. (2001) J. Clin. Invest. 108: 981-990), suggesting that this phosphorylation step may provide a suitable target for anti-cancer therapy. Although the role of p34cdc2-cyclin B1 as a universal mitotic switch is well established (J. Pines (1999) Nat. Cell Biolog. 1: E73-E79), its potential contribution to cell death/survival during spindle checkpoint activation has remained controversial (W. Ongkeko et al. (1995) J. Cell Scie. 108: 2897-2904.; F. Shi et al. (1994) Science 263: 1143-1145).